Fitness costs of Tn1546-type transposons harboring the vanA operon by plasmid type and structural diversity in Enterococcus faecium

Background This study analyzed the genetic traits and fitness costs of vancomycin-resistant Enterococcus faecium (VREfm) blood isolates carrying Tn1546-type transposons harboring the vanA operon. Methods All E. faecium blood isolates were collected from eight general hospitals in South Korea during one-year study period. Antimicrobial susceptibility testing and vanA and vanB PCR were performed. Growth rates of E. faecium isolates were determined. The vanA-positive isolates were subjected to whole genome sequencing and conjugation experiments. Results Among 308 E. faecium isolates, 132 (42.9%) were positive for vanA. All Tn1546-type transposons harboring the vanA operon located on the plasmids, but on the chromosome in seven isolates. The plasmids harboring the vanA operon were grouped into four types; two types of circular, nonconjugative plasmids (Type A, n = 50; Type B, n = 46), and two types of putative linear, conjugative plasmids (Type C, n = 16; Type D, n = 5). Growth rates of vanA-positive E. faecium isolates were significantly lower than those of vanA-negative isolates (P < 0.001), and reduction in growth rate under vancomycin pressure was significantly larger in isolates harboring putative linear plasmids than in those harboring circular plasmids (P = 0.020). Conclusions The possession of vanA operon was costly to bacterial hosts in antimicrobial-free environment, which provide evidence for the importance of reducing vancomycin pressure for prevention of VREfm dissemination. Fitness burden to bacterial hosts was varied by type and size of the vanA operon-harboring plasmid. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-024-00722-2.


Background
Enterococci have emerged as one of the leading causes of hospital-associated bacterial infections due to both intrinsic and acquired resistance against many groups of antimicrobials and tolerance to stresses such as disinfectants.Enterococcus faecalis caused three-fourths of cases of enterococcal infection in humans in the late 1990s [1].However, it has repeatedly been reported in recent years that Enterococcus faecium exceeds E. faecalis in prevalence of human infections, which might be due to rapid adaptation of E. faecium to nosocomial conditions by acquiring resistance against anti-enterococcal antimicrobials, including ampicillin, high-level aminoglycosides, and glycopeptides [2].Furthermore, resistance to glycopeptides in E. faecium has been shown to be an important risk factor for an increased early mortality rate and prolonged hospital stays in patients with bloodstream infections [3,4].
The most common mechanism of resistance against glycopeptides in E. faecium is acquisition of an operon harboring van genes.Among the nine van genotypes, vanA (80-90%) and vanB (10-20%) have been predominantly identified in vancomycin-resistant E. faecium (VREfm), though the proportion of vanA and vanB genes varies geographically [5,6].The vanA operon is composed of regulatory genes (vanR and vanS), genes for peptidoglycan modification enzymes (vanH, vanA, and vanX), and accessory genes (vanY and vanZ) as a part of Tn1546-type transposons located on plasmids [7].The vanB operon includes genes homologous with the vanA operon, except that the vanW gene instead of vanZ is present [8], and it has been commonly identified in Tn1549 or Tn5382 on the bacterial chromosome [9,10].
VREfm of sequence type 17 (ST17) harboring a plasmid with resistance determinant against glycopeptide (VR-plasmid) carrying the vanA operon, has been identified as a major clone showing global dissemination; however, regional distribution of VREfm among diverse STs by country or region and shifts in predominating clones over time by emerging successful clones have also been identified.In Australia, exchange of the dominant vanB-ST796 VREfm clone by vanA-ST1421 has been observed since 2016 [11,12].In Germany, rapid dissemination of the vanB-ST117 VREfm clone resulted in an inversion in prevalence between vanA and vanB (from 2:1 to 1:3) in 2019 [13].In South Korea, vanA-ST17 was the predominating VREfm clone since its emergence in the 1990s, but dissemination of emerging vanA-ST1421 has recently been reported [14].
Shifts in clonal distribution of VREfm might be affected by multiple factors, including environmental factors such as infection control strategies, antimicrobial pressure, and microbial factors including fitness costs of resistance determinants to bacterial hosts [15,16].The fitness cost of acquisition of a plasmid may vary according to the size and replicon type, number and mechanism of resistant alleles, and traits of bacterial hosts [15]; interactions between a plasmid and a bacterial host may also play an important role in determining the cost [16].In general, possession of a plasmid with resistance determinant may be costly, though it could be nearly cost-free or even beneficial to the bacterial host in some cases [17,18].Although fitness costs may be an important factor for emerging successful multidrug-resistant clones, studies on epidemic clones of VREfm are still scarce.
This study was performed to determine the genetic traits of successful VREfm clones in South Korea and their plasmids harboring the vanA operon.Determination of the growth dynamics of VREfm blood isolates was also performed to measure the fitness costs of vanA operon-containing plasmids for bacterial hosts, which might have an effect on shifts in the clonal distribution of VREfm strains.

Study design
All patients with E. faecium bloodstream infection (BSI) between January and December 2019 in eight general hospitals participating in the Global Antimicrobial Surveillance System in South Korea, Kor-GLASS, were included in this study [2].Clinical information, including demographic conditions, underlying comorbidities, and antimicrobial treatment regimens, was investigated.Hospital-originated infection was defined when an initial blood culture was performed after ≥ 2 calendar days of hospitalization.The Charlson comorbidity index and Sepsis-related Organ Failure Assessment (SOFA) score were calculated as previously described [19,20].Clinical outcomes included 30-day mortality, 60-day mortality, and in-hospital mortality.The first E. faecium blood isolate from each patient was collected for microbiological studies, and duplicate isolates were discarded.

Microbiological assessment
Bacterial species were identified using a Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and confirmed by 16S rRNA sequencing.Antimicrobial susceptibility against ampicillin, ciprofloxacin, tetracycline, and quinupristin/dalfopristin was determined by the disk diffusion method.The minimum inhibitory concentrations (MICs) of vancomycin, teicoplanin, linezolid, gentamicin, and streptomycin were determined using the broth microdilution method.Interpretation of zone diameter and MICs were followed the clinical breakpoints of CLSI guideline [21].vanA and vanB gene carriage was evaluated by PCR for all E. faecium isolates [22,23].

Whole-genome sequencing
Genomic DNA was extracted from 132 vanA-positive E. faecium isolates with a GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO).Libraries were prepared using SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences of California, Menlo Park, CA).Entire genomes were sequenced using SMRT cell 1 M by the PacBio Sequel II system (Pacific Biosciences of California).Genome assemblies were performed using PBMM2 (https:// github.com/ Pacifi cBio scien ces/ pbmm2; last updated in June 2021), and annotation of the assembled contigs was performed using PROKKA [24].

Conjugation
Broth mating was performed to estimate the plasmid transfer frequency of vanA operon-harboring plasmids in E. faecium isolates using E. faecium DSM13589 as a recipient.Mixtures of equal amounts of donor and recipient bacterial cells were incubated in Mueller-Hinton (MH) broth (Difco Laboratories, Detroit, MI) and spread on MH agar (Difco Laboratories) containing fusidic acid (20 mg/L), rifampicin (30 mg/L), and vancomycin (4 mg/L).Putative transconjugant cells were confirmed by antimicrobial susceptibility phenotype and possession of vanA, and the conjugation efficiency was calculated per both the number of donor cells and recipient cells.

Bacterial growth rate
Growth rates of the E. faecium isolates were determined by measuring optical density at 600 nm (OD 600 ) using a Multiscan spectrophotometer (Thermo Fisher, Waltham, MA).Bacterial colonies were incubated overnight in Luria-Bertani broth (Difco Laboratories) at 37 °C with shaking, and diluted bacterial suspensions were incubated in MH broth while measuring OD 600 every 3 min.Growth rates of VREfm isolates under both 4 mg/L and 16 mg/L concentrations of vancomycin in MH broth were also determined.Each measurement was replicated three times in the same run, and three independent runs were performed.The average of the maximum slope values of lnOD 600 over time was calculated as the growth rate of the bacterial isolates.

Statistical analysis
Statistical analyses were performed using R software version 4.3.1 (R development Core Team 2023; http:// www.R-proje ct.org/), and the results with P value < 0.05 were considered to be significant.Differences between two groups were analyzed using the Mann-Whitney U test and Fisher's exact test for continuous variables and categorical variables, respectively.Kruskal-Wallis tests were conducted to determine differences among more than three groups, and significant results were further analyzed using post hoc Dunn's tests with Bonferroni correction for pairwise comparisons to identify specific groups with significant differences.A Kaplan-Meier curve was constructed, and the log-rank test was performed.Packages 'ggpubr' and 'ggsurvplot' were used for visualization of the statistical analysis results.

Characteristics of the patients with E. faecium BSI
During the one-year study period, blood culture was performed for 87,399 patients with suspected BSI in eight sentinel hospitals, and 10,990 (12.6%) were positive for at least one bacterial or fungal pathogen.Among them, 308 cases (2.8% of positive blood culture) of E. faecium BSI were identified and included in this study (Table 1).The median age of the patients was 72.5 years, ranging from 61 to 80 years, and more than half (54.9%, 169/308) were male.Most patients were inpatients in general wards (51.6%, n = 159) and intensive care units (37.0%, n = 114); only 11.4% (n = 35) of them were outpatients.Almost four-fifths (77.9%, n = 240) of cases were hospital-originated infections.The most common underlying comorbidities were malignancies (28.2%, n = 87), followed by diabetes mellitus (16.9%, n = 52) and cardiovascular diseases (16.2%, n = 50).

Antimicrobial resistance phenotypes of E. faecium blood isolates
Among the 308 E. faecium blood isolates, 132 (42.9%) showed positive results in vanA PCR, with none being  S1).

Structure of Tn1546-type transposons and glycopeptide resistance phenotypes
vanA operons were identified as a part of Tn1546-type transposons, and 128 blood isolates exhibiting a VanA or VanD phenotype carried one (n = 119) or two (n = 9) copies of the transposon classified as six structural variants both by deletion or truncation of vanY and vanZ and by insertion of ISEfa11 between vanX and IS1216 (Fig. 2A).
The vanS gene of all 128 isolates showed nucleotide sequence variations resulting in three amino acid substitutions, L50V, E54Q, and Q69H, compared with the vanS gene of pIP501 [7,30].The remaining four isolates with vanA + VS phenotypes were found to carry a structurally impaired vanA operon, either lacking one or both regulatory genes vanR and vanS or having a truncated D-alanyl-D-alanine dipeptidase gene vanX, on a plasmid.The 128 isolates with the VanA or VanD phenotype were grouped according to the vanA operon copy number and structure.Group 1 isolates (n = 24) showing the typical VanA phenotype carried a plasmid harboring a copy of a Tn1546-type transposon (Tn1546 vanRSHAXYZ ) with all seven component genes of the vanA operon, with (variant Ib, n = 2) or without (variant Ia, n = 22) insertion of ISEfa11.Group 2 isolates (n = 89) carried a copy of the Tn1546 variant with both truncation of vanY and deletion of vanZ by insertion of an IS1216 into vanY (Tn1546 vanY::IS1216,ΔvanZ ) with (variant IIb, n = 45) or without (variant IIa, n = 44) insertion of ISEfa11.The isolates exhibited VanA or VanD phenotype, and the Tn1546 vanY::IS1216,ΔvanZ variants were found to be located either on a plasmid (n = 85) or on the chromosome (n = 4).The length of remnant vanY in Tn1546 vanY::IS1216,ΔvanZ varied from 165 to 901 bp according to the insertion site, which did not show any correlation with teicoplanin MICs.Group 3 isolates (n = 6) exhibiting the VanD phenotype carried a copy of the Tn1546-type transposon with deletion of both vanY and vanZ [Tn1546 Δ(vanY-vanZ) ] with (variant IIIb, n = 2) or without (variant IIIa, n = 4) insertion of ISEfa11 on a plasmid.Nine isolates harbored two copies of Tn1546-type transposons on a plasmid and/or on the chromosome (Fig. 2B).Of them, five and one isolates exhibiting the VanA phenotype possessed one each copy of Tn1546 vanRSHAXYZ and Tn1546 Δ(vanY-vanZ) and Tn1546 vanY::IS1216,ΔvanZ and Tn1546 Δ(vanY-vanZ) , respectively.The remaining three isolates carried two copies of Tn1546 vanY::IS1216,ΔvanZ and showed the VanA (n = 2) or VanD (n = 1) phenotype (Fig. 2B).

Chromosomal vanA operon
One (n = 6) or two (n = 1) copies of the vanA operon were identified on the chromosome in seven isolates of CT6555-ST1421 (n = 4), CT6141-ST1421 (n = 1), CT6552-ST1421 (n = 1), and CT6557-ST78) (n = 1), and two of them carried an additional vanA operonharboring plasmid (Fig. 3).The vanA operons found on the chromosome were always identified in an insertion unit with (n = 6) or without (n = 2) plasmid-originated components, resulting in variable sizes of the units, ranging from 9 to 43 kb.All eight insertion units were shown to be flanked by a pair of IS1216 of the same or opposite orientations and left (5'-GGT TCT GTT GCA AAG TTT TAA ATC TAC TAT CAA ATA AGG TAG AAT AG-3') and right (5'-GGT TCT GTT GCA AAG TTT TAA ATA AAG AAT AAA ATC CTT ACG GTA TCT AT-3') inverted repeats.The insertion events on the chromosome were shown to be neither site-specific nor nucleotide sequence-specific, occurring in coding regions (n = 5) or in intergenic regions (n = 3).Of note, both isolates C0019EM0016 and C0019EM0037 of CT6555-ST1421 recovered in a hospital with a 7-month gap shared an insertion unit at the same location on the chromosome, indicating a clone; however, the isolate C0019EM0037 carried another insertion unit at a different location on the chromosome, suggesting that another independent insertion event occurred.

VR-plasmids harboring the vanA operon
A total of 127 VR-plasmids carrying one (n = 122) or two (n = 5) copies of Tn1546-type transposons were identified.Circular plasmids of the Inc18 family (n = 96) were most common, followed by putative linear plasmids of the RepB family (n = 21); a circular plasmid of hybrid Inc18:RepA_N was also identified.The remaining nine plasmids were nontypeable due to failure in plasmid All circular plasmids of the Inc18 family were found to share 14-15 kb-sized derivatives from the plasmid pRE25, including a rep2 replication origin, the erm(B) gene, and a zeta-epsilon toxin-antitoxin system, but to lack the probable conjugation regions (ORF25 to ORF39 of pRE25) [31], compatible with the unsuccessful results in conjugation experiments for all E. faecium isolates carrying the plasmids.The 96 circular plasmids were divided into types A (n = 50) and B (n = 46) according to both sequence similarity (> 60%) and Tn1546 variant type (Fig. 4A and B).Type A plasmids had a median size of 32,082 bp, ranging from 18,701 bp to 43,334 bp, and were most frequently identified in E. faecium isolates of ST17 (46%, 23/50).Type A plasmids possessed either Tn1546 variant IIa (n = 45) or variant IIIa (n = 3), except for two plasmids harboring a Tn1546-type transposon A putative replication origin of the RepB family was found in 21 putative linear plasmids with either one (type C, n = 16) or two (type D, n = 5) hairpin ends composed of inverted tandem repeat sequences of 2 kb with 5'-TATA-3' hairpin loops (Fig. 4C and D).The plasmids exhibited homology of approximately 70% with the linear plasmid pELF1 [32], and they shared putative transfer-related components ftsK and parA.More than half (57.1%, 12/21, 10 type C and two type D) of the plasmids were successfully conjugated to the recipient E. faecium DSM13589.The type C plasmids had a median size of 106,938 bp, ranging from 72,306 to 112,181 bp.The single hairpin end of the plasmids were observed to harbor a copy of Tn1546 vanRSHAXYZ (14/16), Tn1546 Δ(vanY-vanZ) (n = 1), or a Tn1546-type transposon with a structurally impaired vanA operon (n = 1); other resistance determinants ant(9)-Ia (n = 6) and erm(A) (n = 5) were also identified at the opposite end of the plasmids.The type D plasmids had a median size of 118,791 bp, ranging from 102,459 to 161,239 bp, and harbored two copies of the
The growth rates of four vanA + VS isolates (median value, 0.200; range, 0.156 to 0.220) were similar to those of vanA-negative isolates.Growth rates of vanA-positive isolates did not differ by strain type or R plasmid type carrying the vanA operon (Fig. 5B and C).Addition of vancomycin at concentrations of 4 mg/L and 16 mg/L to MH broth resulted in a significant decrease in the growth rates of vanA-positive E. faecium isolates, regardless of plasmid type (Fig. 5D-F).Notably, the growth rates of ST1421 and ST17 vanA-positive E. faecium isolates at a vancomycin concentration of 4 mg/L (median value, 0.154; 1st to 3rd interquartile range, 0.139-0.166)were significantly faster than those of vanA-positive isolates of other STs (median value, 0.143; 1st to 3rd interquartile range, 0.122-0.160;Fig. 5G).The difference between growth rates in MH broth without vancomycin and with vancomycin at a concentration of 4 mg/L was significantly lower for isolates carrying a type A or B circular plasmid than those carrying a type C or D linear plasmid (Fig. 5H; P = 0.020).

Discussion
Our experiments revealed a significant difference in growth rates between vanA-positive and vanA-negative E. faecium blood isolates in MH broth, suggesting that it is costly to bacterial hosts in antimicrobial-free environments to carry the vanA operon, regardless of the location of the operon on a plasmid or the chromosome.This fitness burden might be attributed to the basal expression level of the vanA gene even in antimicrobial-free environments [33].The growth rates of four vanA + VS isolates with a structurally impaired vanA operon were similar to those of vanA-negative isolates in our study, though a statistically significant difference with those of VREfm isolates was not observed due to the small number of cases.Complete inactivation of the vanA operon in vanA + VS isolates might be a way to overcome the fitness burden for vanA-carrying E. faecium bacterial hosts when exposed to antimicrobial-free environments [34].These findings provide evidence for the importance of reducing vancomycin pressure through antimicrobial stewardship in clinical fields to prevent dissemination of VREfm.
Addition of vancomycin at a sub-MIC concentration of 4 mg/L to MH broth significantly slowed the growth rates of VREfm isolates compared with those in MH broth without antimicrobials.Both increased fitness burden to bacterial hosts by increased expression level of the vanA gene and growth inhibition effects of vancomycin might affect the growth rates of VREfm isolates [35].It is noteworthy that the difference in growth However, significant difference was not identified in the subgroup analyses with 32 ST17 strains including 26 with circular plasmids and 6 with putative plasmids due to the limited number of strains belonging to same ST.Further investigation about fitness cost according to the bacterial hosts and their plasmid type should be performed.Bacterial hosts might possess a nonconjugative plasmid harboring the Tn1546-type transposon in two possible ways: (1) loss of essential components for conjugation on the plasmid by genetic recombination events after acquisition of the plasmid by conjugation [36,37] and (2) intracellular mobilization of the Tn1546-type transposon from a conjugative plasmid to a nonconjugative plasmid [38].In our study, most of the type A and B nonconjugative plasmids harbored Tn1546 vanY::IS1216,ΔvanZ , whereas most of the type C and D conjugative plasmids harbored Tn1546 vanRSHAXYZ .Tn1546-type transposons were always found to be bracketed by IS1216 elements, and both truncation of vanY and deletion of vanZ in types A and B plasmids might result from insertion of IS1216 at the random sequences of vanY of Tn1546 vanRSHAXYZ , leaving variable sizes of vanY remnants.Furthermore, the EFM isolate G0019EM0008 co-carried a type C plasmid harboring Tn1546 vanRSHAXYZ with an additional pRE25like plasmid, sharing a backbone structure with type A plasmids but lacking Tn1546, which might constitute a snapshot before an intracellular mobilization event of the transposon.
Tn1546-type transposons were also identified on the chromosomes of seven VREfm isolates with or without surrounding components of type B circular plasmids, suggesting the origin of the transposons.Insertion of plasmid-originated antimicrobial resistance determinants into the chromosome has also been reported for other bacterial species, such as ISEcp1-bla CTX-M in Escherichia coli and Salmonella species, and ISAba1bla OXA-23 in Acinetobacter baumannii [39][40][41].This phenomenon might indicate an internalization process of antimicrobial resistance determinants by bacterial hosts in response to constant antimicrobial pressure.Notably, most VREfm isolates harboring the Tn1546-type transposon on their chromosome were shown to belong to ST1421.The ST1421 VREfm isolates, which were first identified in Australia, had a large chromosomal inversion resulting in deletion of 3.5-to 8.7-kb chromosomal sequences, including the pstS gene [42].The high genome plasticity of this notorious clone might be a preferred condition for insertion of the Tn1546-type transposons on the chromosome.
Tn1546 vanY::IS1216,ΔvanZ was found to confer variable levels of resistance against teicoplanin to bacterial hosts, from reduced susceptibility (MIC = 8 mg/L) to high-level resistance (MIC ≥ 32 mg/L), but Tn1546 vanRSHAXYZ conferred high-level resistance, consistent with previous reports [43,44].However, identical nucleotide sequence variations in vanS were identified in all VREfm isolates regardless of their susceptibility phenotype to teicoplanin, inconsistent with a previous report [30], indicating that they might not cause functional changes in VanS protein capability.
A limitation of this study is that the VREfm isolates were collected in a single country, South Korea, and the local distribution of the strain type of VREfm isolates, type of plasmids carrying the Tn1546-type transposon, and structure of the Tn1546-type transposon might be reflected in the results of this study.Another limitation is that the growth rate of E. faecium blood isolates were determined at nutrient-rich conditions, therefore, further investigation including competitive growth and in vivo adaptation experiments and should be performed to clarify the effects of fitness costs.

Conclusions
The possession of Tn1546-type transposon harboring vanA operon was costly to bacterial hosts in antimicrobial-free environment, which provide evidence for the importance of reducing vancomycin pressure for prevention of VREfm dissemination through antimicrobial stewardship in clinical fields.Fitness burden to bacterial hosts of Tn1546-type transposon was varied by type and size of the vanA operon-harboring plasmid, which could have an impact on successful dissemination of the epidemic clones.

Fig. 2
Fig. 2 Structure of Tn1546-type transposons and teicoplanin MICs of E. faecium hosts according to the structure of vanY.Blue bars in the bar graph (C) indicate the clinical breakpoints according to the CLSI guideline, and red one indicates those according to the EUCAST

Fig. 3
Fig. 3 Structure and location of the chromosomal vanA operon.The red bars in chromosome indicate the insertion sites of Tn1546-type transposon.Red arrows indicate the genes identified as resistance determinants, and blue arrows indicate the insertion sequences

Fig. 4
Fig. 4 Structure of four types of plasmids containing Tn1546-type transposons

Table 2
Characteristics of plasmids harboring the vanA operon

pRE25-like type A (n = 50) pRE25-like type B (n = 46) pELF1-like type C (n = 16) pELF1-like Type D (n = 5)
rate was significantly larger in isolates carrying a type C or D putative linear conjugative plasmid (approximately 100-200 kb) than in those carrying a type A or B circular nonconjugative plasmid (approximately 30-40 kb), indicating that the former plasmids are costlier to bacterial hosts under vancomycin pressure.Furthermore, both predominant VREfm clones of ST1421 and ST17, which mostly harbored a type A or B plasmid, exhibited significantly faster growth rates in MH broth with vancomycin than other VREfm isolates.These findings evidence the reason for the success of predominant VREfm clones in hospital settings under persistent vancomycin pressure.